Arenaviruses are the etiological agents of acute hemorrhagic fevers with high mortality in humans. Old World arenaviruses, such as Lassa virus, and New World arenaviruses, such as Junin virus, are transmitted through casual or aerosol contact. Prophylactic vaccines and effective antiviral treatments are urgently needed to curb recurring and emerging outbreaks throughout the world, and to protect the public in the event of a biological attack. Arenaviruses are enveloped viruses, and their envelope glycoproteins, GP-1 and GP-2, mediate receptor binding and membrane fusion. Our preliminary analyses suggest that the heptad-repeat regions of the GP-2 ectodomain may form a six-helix bundle structure that represents the fusion-active conformation of the protein. The long-term goal of this application is to develop a detailed understanding of the structure-function relationships of the Junin virus envelope glycoproteins, and to use this understanding to target the viral entry process for vaccine design and antiviral drug development. Specific aims of this developmental research application are: 1) To characterize the biogenesis and biochemical properties of the Junin virus envelope glycoproteins and to identify the genetic determinants of pH-dependent membrane fusion activity. Scanning mutagenesis will be used to define amino acid residues within the heptad-repeat regions that underlie formation of the fusion-active six-helix bundle. 2) To characterize pH-dependent conformational changes in the GP-1/GP-2 complex using biochemical and immunochemical approaches, in order to understand the acid-induced activation of Junin virus membrane fusion in endosomes. 3) To test whether the envelope glycoprotein-mediated cell-cell fusion assay faithfully recapitulates the early events of Junin virus infection, in an effort to elucidate the mechanism of Junin virus entry and to facilitate the development of protective strategies against arenavirus infection. This application is submitted in response to PA-03-080 for Biodefense and Emerging Infectious Disease Research. [unreadable] [unreadable]